Trinitrotoluene (“TNT”) is a common nitroaromatic compound contaminating the surface and subsurface soil of many military sites. This yellow-colored solid is sometimes used as a reagent in chemical synthesis, but it is best known as a useful explosive material with convenient handling properties.
TNT is poisonous, and skin contact can cause skin irritation, causing the skin to turn a bright yellow-orange color. During the First World War, munition workers who handled the chemical found that their skin turned bright yellow, which resulted in their acquiring the nickname “canary girls” or simply “canaries.” Consumption of TNT produces red urine through the presence of breakdown products and not blood as sometimes believed. Anemia, abnormal liver functions, spleen enlargement and other harmful effects on the immune system have also been found in animals that ingested or breathed trinitrotoluene. There is evidence that TNT adversely affects male fertility and it is listed as a possible human carcinogen. Thus, the toxicity and mutagenicity of TNT has been widely known for decades.
Typically, TNT contamination has been treated by incineration. However, due to the high cost of incineration treatments and the toxicity of incineration products, other methods of decontamination are needed. Bioremediation has become a promising and cost-effective decontamination measure. Indeed, a variety of aerobic and anaerobic bacteria, and even fungi and plants can degrade TNT. When compared to incineration, the previously most common method of biodegradation, bioremediation is both less expensive and less environmentally hazardous.
Bioremediation of TNT by some aerobic microorganisms is problematic, however, because many common degradation pathways lead to the formation of toxic and mutagenic degradation intermediates. TNT consists of a benzene ring, a methyl group, and three nitro groups. When degradation occurs by reducing these nitro groups in a step-wise fashion, the aromatic ring is left uncleaved. These degradation intermediates have been shown to be toxic and/or mutanagenic. Anaerobic degradation pathways, in contrast, avoid these toxic intermediates. Proposed mechanisms for anaerobic TNT metabolism in bacteria are shown in FIG. 6.
The Clostridia are an example of obligate anaerobes that can degrade TNT. C. acetobutylicum is known to enzymatically reduce TNT primarily through the activity of an iron-only hydrogenase, which transfers electrons to TNT through an iron-sulfur center. Related microorganisms reported to anoxically degrade or transform TNT are listed below:
MicroorganismMetabolismClostridium acetobutylicumReduction of TNT to TATClostridium bifermentansDegrades TNT to aliphatic polarCYS-1compounds via 4ADNT and 2,4DANTClostridium bifermentansTransforms TNT into TAT and phenolicLJP-1compoundsClostridium pasterianumReduction of TNT to TATClostridium sordeliiReduction of TNT to TATClostridium sp.Bamberger rearrangement of dihydroxyl-aminonitrotolueneDesulfovibrio sp. strain BTNT as nitrogen source, toluene as aputative intermediateDesulfovibrio sp.TNT as the sole nitrogen source;reduction of TNT to TATDesulfovibrio sp.Transforms TNT into TAT and DANT;42% of radioactivity from [14C]TNT isassociated with cell biomassEscherichia coliReduction of TNT to TATLactobacillus sp.Reduction of TNT to TATMethanococcus sp. strain BReduction of TNT to DANTPseudomonas sp. strainTNT as nitrogen source; TNT as finalJLR11electron acceptorVeillonella alkalescensReduction of TNT to TAT
While studying the degradation of TNT by Clostridium acetobutylicum, an important goal in and of itself, we serendipitously discovered a general method that can be used to drive the production of longer carbon feedstock chemicals in anaerobes having an iron-based hydrogenase. Such methods have value in the art because longer chain molecules can be made into a wider variety of chemicals and because many organisms preferentially produce metabolites having fewer carbons.